Plasmonics nanorod biosensor for in situ intracellular detection of gene expression biomarkers in intact plant systems.

The intracellular developmental processes in plants, particularly concerning lignin polymer formation and biomass production are regulated by microRNAs (miRNAs). MiRNAs including miR397b are important for developing efficient and cost-effective biofuels. However, traditional methods of monitoring miRNA expression, like PCR, are time-consuming, require sample extraction, and lack spatial and temporal resolution, especially in real-world conditions. We present a novel approach using plasmonics nanosensing to monitor miRNA activity within living plant cells without sample extraction. Plasmonic biosensors using surface-enhanced Raman scattering (SERS) detection offer high sensitivity and precise molecular information. We used the Inverse Molecular Sentinel (iMS) biosensor on unique silver-coated gold nanorods (AuNR@Ag) with a high-aspect ratio to penetrate plant cell walls for detecting miR397b within intact living plant cells. MiR397b overexpression has shown promise in reducing lignin content. Thus, monitoring miR397b is essential for cost-effective biofuel generation. This study demonstrates the infiltration of nanorod iMS biosensors and detection of non-native miRNA 397b within plant cells for the first time. The investigation successfully demonstrates the localization of nanorod iMS biosensors through TEM and XRF-based elemental mapping for miRNA detection within plant cells of Nicotiana benthamiana. The study integrates shifted-excitation Raman difference spectroscopy (SERDS) to decrease background interference and enhance target signal extraction. In vivo SERDS testing confirms the dynamic detection of miR397b in Arabidopsis thaliana leaves after infiltration with iMS nanorods and miR397b target. This proof-of-concept study is an important stepping stone towards spatially resolved, intracellular miRNA mapping to monitor biomarkers and biological pathways for developing efficient renewable biofuel sources.

Acoustic separation and concentration of exosomes for nucleotide detection: ASCENDx.

Efficient isolation and analysis of exosomal biomarkers hold transformative potential in biomedical applications. However, current methods are prone to contamination and require costly consumables, expensive equipment, and skilled personnel. Here, we introduce an innovative spaceship-like disc that allows Acoustic Separation and Concentration of Exosomes and Nucleotide Detection: ASCENDx. We created ASCENDx to use acoustically driven disc rotation on a spinning droplet to generate swift separation and concentration of exosomes from patient plasma samples. Integrated plasmonic nanostars on the ASCENDx disc enable label-free detection of enriched exosomes via surface-enhanced Raman scattering. Direct detection of circulating exosomal microRNA biomarkers from patient plasma samples by the ASCENDx platform facilitated a diagnostic assay for colorectal cancer with 95.8% sensitivity and 100% specificity. ASCENDx overcomes existing limitations in exosome-based molecular diagnostics and holds a powerful position for future biomedical research, precision medicine, and point-of-care medical diagnostics.

Cascade Amplified Plasmonics Molecular Biosensor for Sensitive Detection of Disease Biomarkers.

Recent advances in molecular technologies have provided various assay strategies for monitoring biomarkers, such as miRNAs for early detection of various diseases and cancers. However, there is still an urgent unmet need to develop practical and accurate miRNA analytical tools that could facilitate the incorporation of miRNA biomarkers into clinical practice and management. In this study, we demonstrate the feasibility of using a cascade amplification method, referred to as the "Cascade Amplification by Recycling Trigger Probe" (CARTP) strategy, to improve the detection sensitivity of the inverse Molecular Sentinel (iMS) nanobiosensor. The iMS nanobiosensor developed in our laboratory is a unique homogeneous multiplex bioassay technique based on surface-enhanced Raman scattering (SERS) detection, and was used to successfully detect miRNAs from clinical samples. The CARTP strategy based on the toehold-mediated strand displacement reaction is triggered by a linear DNA strand, called the "Recycling Trigger Probe" (RTP) strand, to amplify the iMS SERS signal. Herein, by using the CARTP strategy, we show a significantly improved detection sensitivity with the limit of detection (LOD) of 45 fM, which is 100-fold more sensitive than the non-amplified iMS assay used in our previous report. We envision that the further development and optimization of this strategy ultimately will allow multiplexed detection of miRNA biomarkers with ultra-high sensitivity for clinical translation and application.

miRNA probe integrated biosensor platform using bimetallic nanostars for amplification-free multiplexed detection of circulating colorectal cancer biomarkers in clinical samples.

There is a critical need for sensitive and rapid detection technologies utilizing molecular biotargets such as microRNAs (miRNAs), which regulate gene expression and are a promising class of diagnostic biomarkers for disease detection. Here, we present the development and fabrication of a highly reproducible and robust plasmonic bimetallic nanostar biosensing platform to detect miRNA targets using surfaced-enhanced Raman scattering (SERS)-based gene probes called the inverse Molecular Sentinel (iMS). We investigated and optimized the integration of iMS gene probes onto this SERS substrate, achieving ultra-sensitive detection with limits of detection of 6.8 and 16.7 zmol within the sensing region for two miRNA sequences of interest. Finally, we demonstrated the biomedical usefulness of this nanobiosensor platform with the multiplexed detection of upregulated miRNA targets, miR21 and miR221, from colorectal cancer patient plasma. The resulting SERS data are in excellent agreement with PCR data obtained from patient samples and can distinguish between healthy and cancerous patient samples. These results underline the potential of the iMS-integrated substrate nanobiosensing platform for rapid and sensitive diagnostics of cancer biomarkers for point-of-care applications.

Smartphone-Based Device for Colorimetric Detection of MicroRNA Biomarkers Using Nanoparticle-Based Assay.

The detection of microRNAs (miRNAs) is emerging as a clinically important tool for the non-invasive detection of a wide variety of diseases ranging from cancers and cardiovascular illnesses to infectious diseases. Over the years, miRNA detection schemes have become accessible to clinicians, but they still require sophisticated and bulky laboratory equipment and trained personnel to operate. The exceptional computing ability and ease of use of modern smartphones coupled with fieldable optical detection technologies can provide a useful and portable alternative to these laboratory systems. Herein, we present the development of a smartphone-based device called Krometriks, which is capable of simple and rapid colorimetric detection of microRNA (miRNAs) using a nanoparticle-based assay. The device consists of a smartphone, a 3D printed accessory, and a custom-built dedicated mobile app. We illustrate the utility of Krometriks for the detection of an important miRNA disease biomarker, miR-21, using a nanoplasmonics-based assay developed by our group. We show that Krometriks can detect miRNA down to nanomolar concentrations with detection results comparable to a laboratory-based benchtop spectrophotometer. With slight changes to the accessory design, Krometriks can be made compatible with different types of smartphone models and specifications. Thus, the Krometriks device offers a practical colorimetric platform that has the potential to provide accessible and affordable miRNA diagnostics for point-of-care and field applications in low-resource settings.

Plasmonic nanobiosensors for detection of microRNA cancer biomarkers in clinical samples.

MicroRNAs (miRNAs) play an important role in the regulation of biological processes and have demonstrated great potential as biomarkers for the early detection of various diseases, including esophageal adenocarcinoma (EAC) and Barrett's esophagus (BE), the premalignant metaplasia associated with EAC. Herein, we demonstrate the direct detection of the esophageal cancer biomarker, miR-21, in RNA extracted from 17 endoscopic tissue biopsies using the nanophotonics technology our group has developed, termed the inverse molecular sentinel (iMS) nanobiosensor, with surface-enhanced Raman scattering (SERS) detection. The potential of this label-free, homogeneous biosensor for cancer diagnosis without the need for target amplification was demonstrated by discriminating esophageal cancer and Barrett's esophagus from normal tissue with notable diagnostic accuracy. This work establishes the potential of the iMS nanobiosensor for cancer diagnostics via miRNA detection in clinical samples without the need for target amplification, validating the potential of this assay as part of a new diagnostic strategy. Combining miRNA diagnostics with the nanophotonics technology will result in a paradigm shift in achieving a general molecular analysis tool that has widespread applicability for cancer research as well as detection of cancer. We anticipate further development of this technique for future use in point-of-care testing as an alternative to histopathological diagnosis as our method provides a quick result following RNA isolation, allowing for timely treatment.

Direct and Label-Free Detection of MicroRNA Cancer Biomarkers using SERS-Based Plasmonic Coupling Interference (PCI) Nanoprobes.

MicroRNAs (miRNAs), small noncoding endogenous RNA molecules, are emerging as promising biomarkers for early detection of various diseases and cancers. Practical screening tools and strategies to detect these small molecules are urgently needed to facilitate the translation of miRNA biomarkers into clinical practice. In this study, a label-free biosensing technique based on surface-enhanced Raman scattering (SERS), referred to as plasmonic coupling interference (PCI), was applied for the multiplex detection of miRNA biomarkers. The sensing mechanism of the PCI technique relies on the formation of a nanonetwork consisting of nanoparticles with Raman labels located between adjacent nanoparticles that are interconnected by DNA duplexes. Because of the plasmonic coupling effect of adjacent nanoparticles in the nanonetwork, the Raman labels exhibit intense SERS signals. Such effect can be modulated by the addition of miRNA targets of interest that act as inhibitors to interfere with the formation of this nanonetwork, resulting in a diminished SERS signal. In this study, the PCI technique is theoretically analyzed, and the multiplex capability for detection of multiple miRNA cancer biomarkers is demonstrated, establishing the great potential of PCI nanoprobes as a useful diagnostic tool for medical applications.

Plant cell-surface GIPC sphingolipids sense salt to trigger Ca2+ influx.

Salinity is detrimental to plant growth, crop production and food security worldwide. Excess salt triggers increases in cytosolic Ca2+ concentration, which activate Ca2+-binding proteins and upregulate the Na+/H+ antiporter in order to remove Na+. Salt-induced increases in Ca2+ have long been thought to be involved in the detection of salt stress, but the molecular components of the sensing machinery remain unknown. Here, using Ca2+-imaging-based forward genetic screens, we isolated the Arabidopsis thaliana mutant monocation-induced [Ca2+]i increases 1 (moca1), and identified MOCA1 as a glucuronosyltransferase for glycosyl inositol phosphorylceramide (GIPC) sphingolipids in the plasma membrane. MOCA1 is required for salt-induced depolarization of the cell-surface potential, Ca2+ spikes and waves, Na+/H+ antiporter activation, and regulation of growth. Na+ binds to GIPCs to gate Ca2+ influx channels. This salt-sensing mechanism might imply that plasma-membrane lipids are involved in adaption to various environmental salt levels, and could be used to improve salt resistance in crops.

Inverse Molecular Sentinel-Integrated Fiberoptic Sensor for Direct and in Situ Detection of miRNA Targets.

Molecular advances have been made in analysis systems for a wide variety of applications ranging from biodiagnostics, biosafety, bioengineering, and biofuel research applications. There are, however, limited practical tools necessary for in situ and accurate detection of nucleic acid targets during field work. New technology is needed to translate these molecular advances from laboratory settings into the real-life practical monitoring realm. The exquisite characteristics (e.g., sensitivity and adaptability) of plasmonic nanosensors have made them attractive candidates for field-ready sensing applications. Herein, we have developed a fiber-based plasmonic sensor capable of direct detection (i.e., no washing steps required) of nucleic acid targets, which can be detected simply by immerging the sensor in the sample solution. This sensor is composed of an optical fiber that is decorated with plasmonic nanoprobes based on silver-coated gold nanostars (AuNS@Ag) to detect target nucleic acids using the surface-enhanced Raman scattering (SERS) sensing mechanism of nanoprobes referred to as inverse molecular sentinels (iMS). These fiber-optrodes can be reused for several detection-regeneration cycles (>6). The usefulness and applicability of the iMS fiber-sensors was tested by detecting target miRNA in extracts from leaves of plants that were induced to have different expression levels of miRNA targets. These fiber-optrodes enable direct detection of miRNA in plant tissue extract without the need for complex assays by simply immersing the fiber in the sample solution. The results indicate the fiber-based sensors developed herein have the potential to be a powerful tool for field and in situ analysis of nucleic acid samples.

Plasmonic Nanoprobes for in Vivo Multimodal Sensing and Bioimaging of MicroRNA within Plants.

Monitoring gene expression within whole plants is critical for many applications ranging from plant biology to agricultural biotechnology and biofuel development; however, no method currently exists for in vivo monitoring of genomic targets in plant systems without requiring sample extraction. Herein, we report a unique multimodal method based on plasmonic nanoprobes capable of in vivo imaging and biosensing of microRNA biotargets within whole plant leaves by integrating three different and complementary techniques: surface-enhanced Raman scattering (SERS), X-ray fluorescence (XRF), and plasmonics-enhanced two-photon luminescence (TPL). The method developed uses plasmonic nanostars, which not only provide large Raman signal enhancement but also allow for localization and quantification by XRF and plasmonics-enhanced TPL, owing to gold content and high two-photon luminescence cross sections. Our method uses inverse molecular sentinel nanoprobes for SERS bioimaging of microRNA within Arabidopsis thaliana leaves to provide a dynamic SERS map of detected microRNA targets while also quantifying nanoprobe concentrations using XRF and TPL. The nanoprobes were observed to occupy the intercellular spaces upon infiltration into the leaf tissues. This report lays the foundation for the use of plasmonic nanoprobes for in vivo functional imaging of nucleic acid biotargets in whole plants, a tool that will revolutionize bioengineering research by allowing the study of these biotargets with previously unmet spatial and temporal resolution, 200 µm and 30 min, respectively.

Shining Gold Nanostars: From Cancer Diagnostics to Photothermal Treatment and Immunotherapy.

Cancer has been a significant threat to human health with more than eight million deaths each year in the world. There is an urgent need to develop novel methods to improve cancer management. Biocompatible gold nanostars (GNS) with tip-enhanced electromagnetic and optical properties have been developed and applied for multifunctional cancer diagnostics and therapy (theranostics). The GNS platform can be used for multiple sensing, imaging and treatment modalities, such as surface-enhanced Raman scattering, two-photon photoluminescence, magnetic resonance imaging and computed tomography as well as photothermal therapy and immunotherapy. GNS-mediated photothermal therapy combined with checkpoint immunotherapy has been found to reverse tumor-mediated immunosuppression, leading to the treatment of not only primary tumors but also cancer metastasis as well as inducing effective long-lasting immunity, i.e. an anticancer 'vaccine' effect.

Plasmonic SERS biosensing nanochips for DNA detection.

The development of rapid, cost-effective DNA detection methods for molecular diagnostics at the point-of-care (POC) has been receiving increasing interest. This article reviews several DNA detection techniques based on plasmonic-active nanochip platforms developed in our laboratory over the last 5 years, including the molecular sentinel-on-chip (MSC), the multiplex MSC, and the inverse molecular sentinel-on-chip (iMS-on-Chip). DNA probes were used as the recognition elements, and surface-enhanced Raman scattering (SERS) was used as the signal detection method. Sensing mechanisms were based on hybridization of target sequences and DNA probes, resulting in a distance change between SERS reporters and the nanochip's plasmonic-active surface. As the field intensity of the surface plasmon decays exponentially as a function of distance, the distance change in turn affects SERS signal intensity, thus indicating the presence and capture of the target sequences. Our techniques were single-step DNA detection techniques. Target sequences were detected by simple delivery of sample solutions onto DNA probe-functionalized nanochips and measuring the SERS signal after appropriate incubation times. Target sequence labeling or washing to remove unreacted components was not required, making the techniques simple, easy-to-use, and cost-effective. The usefulness of the nanochip platform-based techniques for medical diagnostics was illustrated by the detection of host genetic biomarkers for respiratory viral infection and of the dengue virus gene.

Multiplexed Detection of MicroRNA Biomarkers Using SERS-Based Inverse Molecular Sentinel (iMS) Nanoprobes.

MicroRNAs (miRNAs) have demonstrated great promise as a novel class of biomarkers for early detection of various cancers, including breast cancer. However, due to technical difficulties in detecting these small molecules, miRNAs have not been adopted into routine clinical practice for early diagnostics. Thus, it is important to develop alternative detection strategies that could offer more advantages over conventional methods. Here, we demonstrate the application of a "turn-on" SERS sensing technology, referred to as "inverse Molecular Sentinel (iMS)" nanoprobes, as a homogeneous assay for multiplexed detection of miRNAs. This SERS nanoprobe involves the use of plasmonic-active nanostars as the sensing platform. The "OFF-to-ON" signal switch is based on a nonenzymatic strand-displacement process and the conformational change of stem-loop (hairpin) oligonucleotide probes upon target binding. This technique was previously used to detect a synthetic DNA sequence of interest. In this study, we modified the design of the nanoprobe to be used for the detection of short (22-nt) miRNA sequences. The demonstration of using iMS nanoprobes to detect miRNAs in real biological samples was performed with total small RNA extracted from breast cancer cell lines. The multiplex capability of the iMS technique was demonstrated using a mixture of the two differently labeled nanoprobes to detect miR-21 and miR-34a miRNA biomarkers for breast cancer. The results of this study demonstrate the feasibility of applying the iMS technique for multiplexed detection of short miRNAs molecules.

In vivo detection of SERS-encoded plasmonic nanostars in human skin grafts and live animal models.

Surface-enhanced Raman scattering (SERS)-active plasmonic nanomaterials have become a promising agent for molecular imaging and multiplex detection. Among the wide variety of plasmonics-active nanoparticles, gold nanostars offer unique plasmon properties that efficiently induce strong SERS signals. Furthermore, nanostars, with their small core size and multiple long thin branches, exhibit high absorption cross sections that are tunable in the near-infrared region of the tissue optical window, rendering them efficient for in vivo spectroscopic detection. This study investigated the use of SERS-encoded gold nanostars for in vivo detection. Ex vivo measurements were performed using human skin grafts to investigate the detection of SERS-encoded nanostars through tissue. We also integrated gold nanostars into a biocompatible scaffold to aid in performing in vivo spectroscopic analyses. In this study, for the first time, we demonstrate in vivo SERS detection of gold nanostars using small animal (rat) as well as large animal (pig) models. The results of this study establish the usefulness and potential of SERS-encoded gold nanostars for future use in long-term in vivo analyte sensing.

Plasmonics-based SERS nanobiosensor for homogeneous nucleic acid detection.

Developing a simple and efficient nucleic acid detection technology is essential for clinical diagnostics. Here, we describe a new conceptually simple and selective "turn on" plasmonics-based nanobiosensor, which integrates non-enzymatic DNA strand-displacement hybridization for specific nucleic acid target identification with surface-enhanced Raman scattering (SERS) detection. This SERS nanobiosensor is a target label-free, and rapid nanoparticle-based biosensing system using a homogeneous assay format that offers a simple and efficient tool for nucleic acid diagnostics. Our results showed that the nanobiosensor provided a limit of detection of ~0.1nM (200amol) in the current bioassay system, and exhibited high specificity for single nucleotide mismatch discrimination. FROM THE CLINICAL EDITOR: Surface-enhanced Raman scattering (SERS) is a sensitive technique that enhances Raman scattering by molecules adsorbed on rough metal surfaces. The enhancement means that the technique may even detect single molecules. In this article, the authors describe a simple and efficient nucleic acid detection technology using SERS, with "OFF-to-ON" signal switch upon nucleic acid target identification and capture, which provides high sensitivity and specificity for single nucleotide mismatch discrimination. This new technology will be most welcomed in clinical diagnostics.

SERS nanosensors and nanoreporters: golden opportunities in biomedical applications.

This article provides an overview of recent developments and applications of surface-enhanced Raman scattering (SERS) nanosensors and nanoreporters in our laboratory for use in biochemical monitoring, medical diagnostics, and therapy. The design and fabrication of different types of plasmonics-active nanostructures are discussed. The SERS nanosensors can be used in various applications including pH sensing, protein detection, and gene diagnostics. For DNA detection the 'Molecular Sentinel' nanoprobe can be used as a homogenous bioassay in solution or on a chip platform. Gold nanostars provide an excellent multi-modality theranostic platform, combining Raman and SERS with two-photon luminescence (TPL) imaging as well as photodynamic therapy (PDT), and photothermal therapy (PTT). Plasmonics-enhanced and optically modulated delivery of nanostars into brain tumor in live animals was demonstrated; photothermal treatment of tumor vasculature may induce inflammasome activation, thus increasing the permeability of the blood brain-tumor barrier. The imaging method using TPL of gold nanostars provides an unprecedented spatial selectivity for enhanced targeted nanostar delivery to cortical tumor tissue. A quintuple-modality nanoreporter based on gold nanostars for SERS, TPL, magnetic resonance imaging (MRI), computed tomography (CT), and PTT has recently been developed. The possibility of combining spectral selectivity and high sensitivity of the SERS process with the inherent molecular specificity of bioreceptor-based nanoprobes provides a unique multiplex and selective diagnostic modality. Several examples of optical detection using SERS in combination with other detection and treatment modalities are discussed to illustrate the usefulness and potential of SERS nanosensors and nanoreporters for medical applications.

DNA bioassay-on-chip using SERS detection for dengue diagnosis.

A novel DNA bioassay-on-chip using surface-enhanced Raman scattering (SERS) on a bimetallic nanowave chip is presented. In this bioassay, SERS signals were measured after a single reaction on the chip's surface without any washing step, making it simple-to-use and reducing the reagent cost. Using the technique, specific oligonucleotide sequences of the dengue virus 4 were detected.

Multiplex detection of disease biomarkers using SERS molecular sentinel-on-chip.

Developing techniques for multiplex detection of disease biomarkers is important for clinical diagnosis. In this work, we have demonstrated for the first time the feasibility of multiplex detection of genetic disease biomarkers using the surface-enhanced Raman scattering (SERS)-based molecular sentinel-on-chip (MSC) diagnostic technology. The molecular sentinel (MS) sensing mechanism is based upon the decrease of SERS intensity when Raman labels tagged at 3'-ends of MS nanoprobes are physically displaced from the nanowave chip's surface upon DNA hybridization. The use of bimetallic layer (silver and gold) for the nanowave fabrication was investigated. SERS measurements were performed immediately following a single hybridization reaction between the target single-stranded DNA sequences and the complementary MS nanoprobes immobilized on the nanowave chip without requiring target labeling (i.e., label-free), secondary hybridization, or post-hybridization washing, thus shortening the assay time and reducing cost. Two nucleic acid transcripts, interferon alpha-inducible protein 27 and interferon-induced protein 44-like, are used as model systems for the multiplex detection concept demonstration. These two genes are well known for their critical role in host immune response to viral infection and can be used as molecular signature for viral infection diagnosis. The results indicate the potential of the MSC technology for nucleic acid biomarker multiplex detection.

Plasmonic nanoprobes: from chemical sensing to medical diagnostics and therapy.

This article provides an overview of the development and applications of plasmonics-active nanoprobes in our laboratory for chemical sensing, medical diagnostics and therapy. Molecular Sentinel nanoprobes provide a unique tool for DNA/RNA biomarker detection both in a homogeneous solution or on a chip platform for medical diagnostics. The possibility of combining spectral selectivity and high sensitivity of the surface-enhanced Raman scattering (SERS) process with the inherent molecular specificity of nanoprobes provides an important multiplex diagnostic modality. Gold nanostars can provide an excellent multi-modality platform, combining two-photon luminescence with photothermal therapy as well as Raman imaging with photodynamic therapy. Several examples of optical detection using SERS and photonics-based treatments are presented to illustrate the usefulness and potential of the plasmonic nanoprobes for theranostics, which seamlessly combines diagnostics and therapy.

Surface-enhanced Raman scattering molecular sentinel nanoprobes for viral infection diagnostics.

In this paper, we describe a surface-enhanced Raman scattering (SERS)-based detection approach, referred to as "molecular sentinel" (MS) plasmonic nanoprobes, to detect an RNA target related to viral infection. The MS method is essentially a label-free technique incorporating the SERS effect modulation scheme associated with silver nanoparticles and Raman dye-labeled DNA hairpin probes. Hybridization with target sequences opens the hairpin and spatially separates the Raman label from the silver surface thus reducing the SERS signal of the label. Herein, we have developed a MS nanoprobe to detect the human radical S-adenosyl methionine domain containing 2 (RSAD2) RNA target as a model system for method demonstration. The human RSAD2 gene has recently emerged as a novel host-response biomarker for diagnosis of respiratory infections. Our results showed that the RSAD2 MS nanoprobes exhibits high specificity and can detect as low as 1 nM target sequences. With the use of a portable Raman spectrometer and total RNA samples, we have also demonstrated for the first time the potential of the MS nanoprobe technology for detection of host-response RNA biomarkers for infectious disease diagnostics.